Hyperpolarized helium-3 microbubble gas entrapment methods and associated products

ABSTRACT

Methods for increasing the T 1  of injectable microbubble formulations of hyperpolarized  3 He include the step of introducing the hyperpolarized  3 He to a quantity of microbubbles held in a chamber and increasing the pressure therein to facilitate the movement or loading of the  3 He into the microbubbles. Subsequently, a limited quantity of carrier liquid or a carrier liquid solution alone, or pre-mixed with  3 He, can be introduced to the microbubble/ 3 He in the chamber to inhibit the tendency of the  3 He to leach out of the bubble. Related pharmaceutical products and associated containers as well as an evacuation based method for rapid mixing and delivery of the bubbles and the  3 He is also disclosed. An additional method for dissolving  129 Xe gas by using bubbles as an accelerant is also described.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 09/428,814filed Oct. 28, 1999, U.S. Pat. No. 6,284,222, which claims the benefitof priority from provisional U.S. Application Ser. No. 60/106,843, filedNov. 3, 1998. The contents of these documents are hereby incorporated byreference as if recited in full herein.

FIELD OF THE INVENTION

The present invention relates generally to hyperpolarized Helium-3(“³He”) and is particularly suitable for Magnetic Resonance Imaging(“MRI”) and NMR spectroscopic medical diagnostic applications.

BACKGROUND OF THE INVENTION

Conventionally, MRI has been used to produce images by exciting thenuclei of hydrogen molecules (present in water protons) in the humanbody. However, it has recently been discovered that polarized noblegases can produce improved images of certain areas and regions of thebody, which have heretofore produced less than satisfactory images inthis modality. Polarized ³He and Xenon-129 (“¹²⁹Xe”) have been found tobe particularly suited for this purpose. Unfortunately, as will bediscussed further below, the polarized state of the gases are sensitiveto handling and environmental conditions and, undesirably, can decayfrom the polarized state relatively quickly.

“Polarization” or hyperpolarization of certain noble gas nuclei (such as¹²⁹Xe or ³He) over the natural or equilibrium levels, i.e., theBoltzmann polarization, is desirable because it enhances and increasesMRI signal intensity, allowing physicians to obtain better images of thesubstance in the body. See U.S. Pat. No. 5,545,396 to Albert et al., thedisclosure of which is hereby incorporated herein by reference as ifrecited in full herein.

For medical applications, after the hyperpolarized gas is produced, itis processed to form a non-toxic or sterile composition prior tointroduction into a patient. Unfortunately, during and after collection,the hyperpolarized gas can deteriorate or decay (lose its hyperpolarizedstate) relatively quickly and therefore must be handled, collected,transported, and stored carefully. The “T₁” decay constant associatedwith the hyperpolarized gas' longitudinal relaxation time is often usedto describe the length of time it takes a gas sample to depolarize in agiven container. The handling of the hyperpolarized gas is critical,because of the sensitivity of the hyperpolarized state to environmentaland handling factors and the potential for undesirable decay of the gasfrom its hyperpolarized state prior to the planned end use, i.e.,delivery to a patient. Processing, transporting, and storing thehyperpolarized gases—as well as delivery of the gas to the patient orend user—can expose the hyperpolarized gases to various relaxationmechanisms such as magnetic gradients, ambient and contact impurities,and the like.

In the past, various hyperpolarized delivery modes such as injection andinhalation have been proposed to introduce the hyperpolarized gas to apatient. Inhalation of the hyperpolarized gas is typically preferred forlung or respiratory type images. To target other regions, other deliverypaths and techniques can be employed. However, because helium is muchless soluble than xenon in conventional carrier fluids such as lipids orblood, ³He has been used almost exclusively to image the lungs ratherthan other target regions.

Recent developments have proposed overcoming the low solubility problemof helium by using a micro-bubble suspension. See Chawla et al., In vivomagnetic resonance vascular imaging using laser-polarized ³ Hemicrobubbles, 95 Proc. Natl. Acad. Sci. USA, pp. 10832-10835 (September1998). Chawla et al. suggests using radiographic contrast agents as theinjection fluid to deliver microbubbles of hyperpolarized ³He gas in aninjectable formulation. This formulation can then be injected into apatient in order to image the vascular system of a patient.

Generally stated, one way currently used to load or produce themicrobubble mixture is via “passive” permeability. That is, thehyperpolarized ³He typically enters the walls of the micro-bubbles basedon the helium permeability of the bubble itself. Thus, this gas loadingmethod can take an undesirable amount of time, which can allow thehyperpolarized gas to decay unduly. Further, contact with the fluid oreven the microbubble can result in contact-induced depolarization whichcan dominate the relaxation mechanisms of the hyperpolarized ³He andcause an undesirable reduction in the hyperpolarized life of the gas.

As such, there remains a need to improve micro-bubble ³He formulationsand loading methods to minimize the decay of the polarized gas andimprove the T₁ of the micro-bubble formulation.

In addition, there is also a need to increase the ease of solubilizinghyperpolarized gaseous xenon, which, in the past, has been problematic.

OBJECTS AND SUMMARY OF THE INVENTION

It is therefore an object of the present invention to improve the T₁ fora hyperpolarized ³He microbubble injectable solution.

It is another object of the present invention to reduce the effect ofcontact-induced depolarization to increase the hyperpolarized life of aninjectable microbubble product.

It is an additional object of the present invention to produce aninjectable microbubble solution in a way which increases theconcentration of hyperpolarized ³He in the microbubbles in theinjectable formulation.

It is another object of the invention to provide methods and devices foradministering polarized microbubble injectable formulations to a subjectin a manner which can rapidly mix and deliver the formulation tocapitalize on the polarized state of the gas before it deleteriouslydecays.

It is another object of the present invention to process and form ahyperpolarized ³He gas mixture in improved containers and injectiondelivery systems which are configured to inhibit depolarization in thecollected polarized gas.

It is yet another object of the invention to provide methods, surfacematerials and containers which will minimize the depolarizing effects ofthe hyperpolarized state of the ³He gas in a microbubble solutionattributed to one or more of paramagnetic impurities, oxygen exposure,stray magnetic fields, and surface contact relaxation.

It is another object of the present invention to provide a dissolutionassist method for facilitating the transition of hyperpolarized ¹²⁹Xefrom a gaseous to a liquid state.

These and other objects are satisfied by the present invention, which isdirected to microbubble related hyperpolarized gas injectable solution(solubilized or liquid) products and related production and deliverymethods, systems, and apparatus.

A first aspect of the present invention is directed to a method ofproducing an injectable formulation of hyperpolarized ³He. The methodincludes the steps of introducing a plurality of microbubbles into achamber and then directing a quantity of hyperpolarized ³He into thechamber with the plurality of microbubbles. The pressure in thecontainer is increased to above one atmosphere. A quantity of liquid isthen directed into the chamber after the quantity of hyperpolarized gasand the microbubbles are located therein. The microbubbles with the(filled) hyperpolarized ³He contact the liquid thereby producing aninjectable formulation of hyperpolarized ³He microbubbles.

In a preferred embodiment, the pressure is increased to above 2atmospheres, and preferably increased to between about 2-10 atm. It isalso preferred that the increasing step is performed after themicrobubbles are introduced into the chamber and before the liquid isintroduced therein.

Preferably, the liquid solution is selected such that it inhibits thedepolarization of the gas based on contact with same. For example, inone embodiment, the fluid is selected such that it has low solubilityvalues for ³He (preferably less than about 0.01, and more preferablyless than about 0.005-0.008) or high diffusion coefficient value for³He. In operation, the microbubble surface or walls are configured inthe absence of the injection liquid to allow the hyperpolarized ³He tofreely enter through the exterior cage-like shell of the bubble, thenthe fluid or liquid wraps around the openings in the cage-like shell totrap the hyperpolarized gas therein in such a way as to inhibit thetransfer or leaching of the gas out of the microbubble. In addition, oralternatively, the fluid itself is introduced in a relatively limitedquantity which can reduce the pressure differential between the ³He inthe bubbles and those in the fluid and/or a quantity of ³He can bepremixed with the liquid solution. The reduced pressure differential(saturation or equilibrium of the ³He in the fluid external of thebubbles) can reduce the amount of ³He which migrates therefrom.

In addition, even if the ³He exits the bubble, the low solubility of theselected fluid can reduce the amount of migration of helium from thebubble until equilibrium/saturation to prolong polarization associatedtherewith, thereby prolonging the T₁ of the microbubble injectablemixture. Indeed, the selection of the fluid will be an important factorin establishing a sufficiently long T₁ for the injectable formulationitself. Alternatively, or additionally, for formulations directed to ³Hedissolved into liquid, it is preferred that the liquid have a highdiffusion coefficient for ³He (high diffusion preferably meaning about1.0×10⁻⁵ cm²/s and more preferably at least 1.0×10⁻⁴ cm²/s).

Another aspect of the present invention is directed toward a method ofmixing and formulating polarized gaseous ³He for in vivo injection. Themethod includes the steps of introducing a quantity of microbubbles intoa container and applying a vacuum to the container. The method alsoincludes directing a first quantity of hyperpolarized ³He gas into theevacuated container with the microbubbles and directing a secondquantity of a fluid into the container thereafter to form a bubblesolution. The bubble solution is then removed from the container andinjected into a subject.

Preferably, the second quantity of fluid comprises a substantiallydeoxygenated fluid and the injecting step includes delivering the bubblesolution to an in situ positioned catheter inserted into the vein of asubject. It is also preferred that the mixing portion of the method becarried out temporally proximate to the injecting step (preferablyperformed within about 30 seconds prior to the injection).

An additional aspect of the present invention is directed toward amethod of solubilizing gaseous hyperpolarized ¹²⁹Xe. The method includesthe steps of introducing a first quantity of bubbles into a chamber anddirecting a second quantity of hyperpolarized ¹²⁹Xe into the chambersuch that at least a portion of the ¹²⁹Xe contacts the microbubbles. Themethod also includes the steps of dissolving a portion of the ¹²⁹Xe andthen separating substantially all of the microbubbles from the ¹²⁹Xeprior to delivery of the dissolved phase of the 129Xe to a subject. Themicrobubbles act as an accelerant to solubilize the ¹²⁹Xe from a gaseousstate.

Yet another aspect of the present invention is a pharmaceuticalinjectable in vivo fluid hyperpolarized product. The product includes afirst quantity of microbubbles formed from a first material and a secondquantity of hyperpolarized ³He. The product also includes a thirdquantity of a liquid carrier solution. The third quantity is less thanor substantially equal to the sum of the first and second quantities.

Preferably, the microbubbles are sized to be less than about 10 μm indiameter and the injectable product is single bolus sized as about 50cc's.

The present invention includes methods to increase the density of the³He in each microbubble (increasing the loading density) and to increasethe bubble packing density to “pack” the bubbles more densely in thesolution. Each can provide one or more of stronger signal strength andgreater effective T₁'s.

Further, the present invention can allow reduced bolus sized quantitiesof ³He. For example, venous hyperpolarized gas microbubble injectionvolumes of from about 5-50 cc's, and more preferably about 15-30 cc's,can provide sufficient signal for clinically useful images. Preferably,the microbubble formulations of the present invention are also formedsuch that the gas microbubbles are sized to be less than about 10 μm andmore preferably about 8 μm or less in diameter so as to be able to beinjected in a venous side of the circulation system and then passthrough the capillaries to the arterial side of the circulation system

Advantageously, one or more of the loading of the gas into the bubble,and the delay in its escape, and the fluid packing and fluidcompatibility can facilitate the delivery of quantities of the ³He in amanner which can allow the gas to be injected into a target area in asufficient quantity and strength to provide clinically usefulinformation.

The present invention, recognizing the very limited (T₁) life of themicrobubble formulations, also provides a rapid mixing and deliverydevice which can allow the bubble mixing and formulation preparationtemporally proximate to the point of injection (preferably injected viaa catheter). The present invention also allows for an NMR coil to bepositioned on and/or operably associated with the microbubbleformulation (on the gas-filled bubble formulation holding chamber orassociated conduits, catheters, or holding chamber stems and the like)to allow for a polarization measurement to be conveniently obtained inconjunction with a planned delivery to better calibrate the signalintensity and/or reduce the delivery of depolarized substances.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates a microbubble cage-like shell (thespecific configuration and size of the opening(s) in the “shell” beingattributed to the molecular structure of the bubble material itself) andloading method according to the present invention.

FIG. 1A schematically illustrates an alternative embodiment of amicrobubble structure according to the present invention.

FIG. 2 schematically illustrates an apparatus or introduces a liquidinto a microbubble and hyperpolarized ³He gas mixture.

FIG. 3 schematically illustrates the liquid of FIG. 2 forming an outerwall or closing the cage-like opening(s) in the microbubble shell totrap the hyperpolarized gas therein.

FIG. 4 schematically illustrates the microbubble mixture of FIG. 2 beingwithdrawn from the mixing container in preparation of injecting apredetermined amount into a target.

FIG. 5A is a front view schematic illustration of an evacuated deliveryand mixing system.

FIG. 5B is a front view schematic illustration of FIG. 5A showing asyringe (the syringe and container are shown exaggerated in scale forease of representation) withdrawal and injectable delivery technique.

FIG. 6 is a block diagram of a method of formulating a microbubbleinjectable product.

DETAILED DESCRIPTION OF THE INVENTION

The present invention now will be described more fully hereinafter withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown. This invention may, however, be embodied inmany different forms and should not be construed as limited to theembodiments set forth herein; rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art. Likenumbers refer to like elements throughout. In the figures, certainfeatures have been exaggerated for clarity or drawn for illustrationpurposes and as such the figures are not drawn to scale. For example, atypical microbubble is sized such that it is much larger (preferablysized at about 10 μm or less) than a 5 Å atom of ³He (e.g., about 2000times larger). In addition, it will be appreciated that the molecularmake-up of the bubble (corresponding to the particular bubblematerial(s)) will provide the specific configuration and size of theopening(s) and structure of the shell and walls and the figures hereinare merely for schematic representation and discussion.

Background—Polarized Gas Relaxation Processes

Once hyperpolarized, there is a theoretical upper limit on therelaxation time (T₁) of the polarized gas based on the collisionalrelaxation explained by fundamental physics, i.e., the time it takes fora given sample to decay or depolarize due to collisions of thehyperpolarized gas atoms with each other absent other depolarizingfactors. For example, ³He atoms relax through a dipole-dipoleinteraction during ³He—³He collisions, while ¹²⁹Xe atoms relax throughN·I spin rotation interaction (where N is the molecular angular momentumand I designates nuclear spin rotation) during ¹²⁹Xe—¹²⁹Xe collisions.Stated differently, the angular momentum associated with flipping over anuclear spin is conserved by its being taken up by the rotationalangular momentum of the colliding atoms. In any event, because bothprocesses occur during noble gas-noble gas collisions, both resultingrelaxation rates are directly proportional to gas pressure (T₁ isinversely proportional to pressure). Thus, at one atmosphere, thetheoretical relaxation time (T₁) of ³He is about 744-760 hours, whilefor ¹²⁹Xe the corresponding relaxation time is about 56 hours. SeeNewbury et al., Gaseous ³ He— ³ He Magnetic Dipolar Spin Relaxation, 48Phys. Rev. A, No. 6, p. 4411 (1993); Hunt et al., Nuclear MagneticResonance of ²⁹ Xe in Natural Xenon, 130 Phys. Rev. p. 2302 (1963).Unfortunately, other relaxation processes prevent the realization ofthese theoretical relaxation times. For example, the collisions ofgaseous ¹²⁹Xe and ³He with container walls (“surface relaxation”) havehistorically dominated most relaxation processes. For ³He, most of theknown longer relaxation times have been achieved in special glasscontainers having a low permeability to helium. In the past, afundamental understanding of surface relaxation mechanisms has beenelusive, which has made the predictability of the associated T₁difficult.

U.S. Pat. No. 5,612,103 to Driehuys et al. describes using coatings toinhibit the surface-induced nuclear spin relaxation of hyperpolarizednoble gases, especially ¹²⁹Xe. The disclosure of this patent is herebyincorporated by reference as if recited in full herein. Driehuys et al.recognized that nuclear spin relaxation of ¹²⁹Xe on apolydimethoylsiloxane (“PDMS”) surface coating can be dominated bydipolar coupling of the ¹²⁹Xe nuclear spin to the protons in the polymermatrix. Thus, it was demonstrated that paramagnetic contaminants (suchas the presence of paramagnetic molecules like oxygen) were not thedominant relaxation mechanism in that system because the inter-nucleardipole-dipole relaxation was found to dominate the system underinvestigation. This was because ¹²⁹Xe substantially dissolved into theparticular polymer matrix (PDMS) under investigation. See BastiaanDriehuys et al., Surface Relaxation Mechanisms of Laser-Polarized ¹²⁹Xe, 74 Phys. Rev. Lett., No. 24, pp. 4943-4946 (1995).

Background—Relaxivity of Materials

In order to compare the characteristic information of certain materialsconcerning their respective relaxing effects on hyperpolarized noblegases, the term “relaxivity” is used. As used herein, the term“relaxivity” (“Υ”) is used to describe a material property associatedwith the rate of depolarization (“1/T₁”) of the hyperpolarized gassample. See co-pending and co-assigned U.S. patent application Ser. No.09/126,448, entitled Containers for Hyperpolarized Gases and AssociatedMethods, the disclosure of which is hereby incorporated by reference asif fully recited herein.

Generally stated, gas dissolved in the polymer surface relaxes quickly(less than one second), so most of the hyperpolarized gas in thecontainer is in the free gas form. Therefore, relaxation of this gasoccurs through continual exchange between the free gas and the gasdissolved in the polymer. In material quantities, the rate of this gasexchange can be described by the “sorption parameters”—solubility (“S”),diffusion coefficient (“D”), and permeability (“P”). Permeability is thetransmission of atoms or molecules through a (polymer) film. It dependson chemical and physical structure of the material as well the structureand physical characteristics of the permeant molecules. Permeability canbe defined as the product of solubility and the diffusion coefficient(“P=S×D”). Solubility (“S”) is a measure of how much permeant can bedissolved in a given material. Diffusion coefficient (“D”) is a measureof the random mobility of the atoms in the polymer. Thus, the polymersorption parameters can be used to characterize the relaxation ofhyperpolarized gases in the presence of permeable surfaces.

As discussed in the above-referenced patent application, the relaxationrate (“Γ_(p)”) in the polymer terms can be rewritten in terms of T₁,Γ_(p)=1/T_(1p). Solving for the relaxation time T₁: $\begin{matrix}{T_{1} = {\frac{a}{S}\sqrt{\frac{T_{1}^{p}}{D_{p}}}}} & (1.00)\end{matrix}$

This analysis can be extended into three dimensions, yielding:$\begin{matrix}{T_{1} = {\frac{V_{c}}{A_{p}S}\sqrt{\frac{T_{1}^{p}}{D_{p}}}}} & (1.10)\end{matrix}$

where V_(c) is the internal volume of the chamber, A_(p) is the exposedsurface area of the polymer and S is the solubility of the gas in thepolymer.

The inverse relationship between T₁ and S is a key observation from thisdevelopment. There is also an apparent inverse square root dependence onthe diffusion coefficient D_(p). However, the relaxation time in thepolymer 1/T_(p) also depends on D_(p), canceling the overall effect onT₁. This leaves solubility as the dominant sorption characteristic indetermining T₁.

³He Microbubble Relaxation Considerations

The hyperpolarized ³He is introduced to at least three different contactrelated relaxation mechanisms when formulating the suspension mixture:the injection container related parameters such as the size, shape andmaterial (as well as the materials of the proximately located seals andother components) of the container, the microbubble related parameterssuch as size, shape and material, and the injection fluid material. Thecontainer will be discussed further below but is preferably configuredand formed from materials such that it is polarization-friendly.

Generally described, the microbubble acts as a miniature container tohold the hyperpolarized ³He. As such, the gas is preferably introducedinto the “bubble” in a relaxation-efficient manner. Further, thestructure of the microbubble is preferably such that the ³He can freelyenter into the bubble through the exterior walls of the bubble in theabsence of the injection liquid. Still further, the bubble is preferablyloaded such that it retains increased amounts of ³He. The instantinvention provides several alternatives of suitable material structure,and loading methods for the microbubble configurations in combinationwith different injection fluids and preferred associated materialproperty values thereof as they relate to hyperpolarized ³He in order tooptimize the microbubble injectable solution T₁.

In addition, it will be appreciated by one of skill in the art that theshell or wall thickness of the typical bubble is much thinner than thecritical length scale L_(p) (defined and discussed below). For example,for silicone, D=4.1 e⁻⁵ cm²/s and T_(p)=4.5 s, and the associated lengthscale is about 100 μm. In contrast, the wall thickness of a typicalbubble can be 5-6 orders of magnitude thinner (about 100 Å), therebysignificantly reducing the role of the bubble wall in the depolarizationanalysis. Thus, the T₁ of the ³He gas in the bubble is not shorter thanthe T₁ of ³He in the fluid, i.e., it allows for increased or improvedimage acquisition time or more distal target imaging regions from thepoint of injection.

Loading

Assuming the microbubble is a spherical bubble of radius “R” andassociated area (“A”) of the shell with shell thickness “Δx” and thatthe initial volume of gas (V_(g)) which exists in the bubble shell isgiven by the product of gas solubility in the shell material (S), thegas density outside the shell [G]_(o), and the volume of the shell(ΔxA), then

V _(g) =S[G] _(o) ΔxA  (2)

The time it takes for this volume of gas to permeate through to theinterior is limited by diffusion. Nominally, this time is given byt=(Δx²/D).

Thus, the volume of gas permeating into the shell per unit time can beexpressed by: $\begin{matrix}{{\frac{}{t}V_{g}} = \frac{{S\lbrack G\rbrack}_{o}{DA}}{\Delta \quad x}} & (3)\end{matrix}$

A more complex analysis may take into account the build-up of gas on theinterior of the bubble and the resulting differential equation. As theinterior gas concentration builds, the transfer of gas from the exteriorwill slow, and equilibrium will be reached in the form of a chargingcapacitor. However, a good working estimate of how long it takes to“load” the interior of the bubble with polarized ³He can be obtainedwithout this analysis. This loading time can be described as$\begin{matrix}{{\frac{}{t}V_{g}t_{load}} = V} & (4)\end{matrix}$

where (V) is the interior volume of the bubble. Thus, according toequation (5), estimates of hyperpolarized gas loading time into thebubble can be calculated as $\begin{matrix}{t_{load} = \frac{R\quad \Delta \quad x}{3{S\lbrack G\rbrack}_{o}D}} & (5)\end{matrix}$

For example, assuming that R=5 μm=5×10⁻⁴ cm and [G]_(o)=1 amagat and ahelium solubility of S≈0.01 (which it typically is for most preferredmaterials), and using an estimate of a diffusion coefficient of heliumin LDPE which is D=6.8×10⁻⁶ cm²/s and an extremely thin wall Δx=100Å=10⁻⁶ cm, the diffusion time is calculated as a reasonable t≈2.5 ms.For a larger wall thickness, on the order of Δx=1 μm=1.0×10⁻⁴ cm, theloading time increases to 0.25 s, which, although slower, is stillrelatively good.

Increasing the Loading Density

As shown in FIG. 2, another way to increase the T₁ of the microbubblemixture is to increase the density or quantity of ³He loaded into themicrobubble. This increased loading density can be attained byincreasing the pressure in the preparation container to force additionalquantities of ³He into a microbubble 10. The increased density of thepolarized ³He in a microbubble 10 can prolong the effective T₁ of thesolution. For example, escaped ³He can act to equilibrate the solutionsurrounding the microbubble thereby helping to retain partial quantitiesof the hyperpolarized ³He protected/isolated inside the bubble. Anotheradvantage to increasing loading density is that increased quantities of³He in the microbubbles can provide larger signal strength. Preferably,to load, the microbubble spheres are positioned in the container, thehyperpolarized gas is introduced via an air tight seal into thecontainer and the pressure in the container 30 is increased aboveatmospheric pressure, preferably in the range of about 2-8 atm, and morepreferably above 8 atm, and still more preferably up to about 10 atm, tocreate more densely packed bubbles.

Liquid Introduction

As will be appreciated by those of skill in the art, typically amicrobubble which is structurally (at a molecular level) configured toallow the molecular ³He to enter will typically also just as easilyallow it to leave. Thus, it is preferred that, once the hyperpolarizedgas is inside the bubble, either the bubble and/or the liquid mixtureacts to prevent or inhibit the gas from exiting from the interior of thebubble. In a preferred embodiment, the solution or mixture liquid isselected such that if the hyperpolarized gas does exit from the bubble,it contacts liquid which has a low solubility for the ³He, therebylimiting the total quantity of ³He which exits the bubble. “Lowsolubility” includes solutions selected such that they have a solubilityfor ³He (“S”) which is less than about 0.01, and preferably less about0.008, and more preferably less than about 0.005.

Alternatively, for solutions targeted at facilitating ³He dissolved inthe mixture, the liquid can be selected such that the polarized ³He hasa high diffusion coefficient therewith. Preferably, “high diffusion”means diffusion coefficient rates of above 1×10⁻⁶ cm²/s, and preferablymore than about 6×10⁻⁶cm²/s, and still more preferably above about1.0×10⁻⁵ cm²/s, and even more preferably on the order of at least1.0×10⁻⁴ cm²/s. Advantageously, a relatively long T₁ for the solutioncan be achieved for ³He dissolved in fluid by selecting a fluid whichhas a high diffusion coefficient for ³He.

The instant invention recognizes that the injection formulation ofmicrobubbles can be improved by optimizing the microbubble structureitself to provide faster transport of the ³He therein. Preferably, theimproved structure is provided by formulating a substantial quantity ofthe bubbles with a surface contact material which is selected to have alow solubility value for ³He. In an alternative embodiment, themicrobubble material is selected such that it has a relatively thin walland high diffusion coefficient value for ³He, which allows for the ³Heto move into the bubble more rapidly.

Preferably, the bubbles are sized and configured with thin bubble wallthicknesses and miniaturized microbubble diameters. As used herein,“thin” means a bubble wall thickness of less than about 6 microns, andmore preferably a wall thickness of about 1-2 microns. “Miniaturized”includes microbubble diameters which are less than about 10 microns, andpreferably less than about 8 microns. The miniaturized size of thebubbles are particularly preferred for perfusion related images suchthat the bubbles are below about 8 microns so that they can freelytravel into and/or through capillaries.

Referring to FIG. 1, one embodiment of the instant invention recognizesthat a microbubble 10 can advantageously be formed from a material whichis physiologically compatible and has a cage-like structure with walls15 which can allow for quicker transport of the hyperpolarized gas 20into the microbubble 10. The walls 15 of the microbubble define anopening 15 a which is preferably sized such that it is slightly largerthan the ³He. The ³He atom is on the order of about 2 Å-5 Å in diameterand thus the opening 15 a in the wall of the microbubble is preferablylarger than 2 Å. One alternative microbubble embodiment is schematicallyillustrated in FIG. 1A, in which a microbubble 10′ has cage-like walls15′.

In operation, as shown in FIG. 2, the ³He 20 and microbubbles 10 arepositioned in a polarization friendly container and the hyperpolarized³He 20 freely enters into the opening(s) 15 a in the microbubble until astate of substantial equilibrium is reached. Preferably, the transporttime (the time it takes the gas to enter the microbubbles) at increasedpressure (above 1 atm) is below about 1 minute for a single dosageamount. That is, in a preferred embodiment, a quantity of microbubblesis introduced into a properly prepared and air tight sealed container(first). The pressure in the container is then elevated to above 1 atm,preferably to about 2-8 atm, and more preferably to above 8 atm, andstill more preferably to about 10 atm. A quantity of polarized ³He gasis then subsequently directed into the container. The pressurefacilitates the tendency of a portion of the hyperpolarized ³He to enterthe bubble structure. Of course, the pressure can also be increasedduring the introduction of the ³He or even shortly thereafter (or everprior to the introduction of the microbubbles, although not preferred).Preferably, the liquid is also injected into the container while thepressure is elevated. This liquid elevated pressure can be either atsubstantially the same pressure or a reduced elevated pressure from themicrobubble/hyperpolarized gas loading pressure.

Also as shown in FIG. 2, after a predetermined (relatively short)transport time (typically less than about one minute as noted above,i.e., the time for at least a portion of the polarized ³He to move intothe bubble structure), a liquid or fluid 40 is introduced into thecontainer 30. In one preferred embodiment, it is preferred that theliquid 40 be selected such that the ³He has low solubility for the fluid(“S” less than about 0.01, and more preferably less than about 0.008,and still more preferably less than about 0.005). The low solubilityhelps inhibit polarization decay and preferably plugs the gaps oropenings in the microbubble wall such that the ³He 20 is inhibited fromleaving the microbubble. As shown in FIG. 4, the liquid 40 surrounds themicrobubble and because the ³He is substantially insoluble in the fluid,the ³He 20 is repelled by contact with the liquid 40. Further, as shownin FIG. 3, the liquid forms the outer wall 41 of the microbubble 10,thus effectively “trapping” the ³He 20 in the microbubble. Of course, asnoted above, the liquid can also be selected such that it has a highdiffusion coefficient for the ³He. In any event, the ³He 20 andmicrobubbles 10 together with a solution or fluid mixture form aninjectable formulation which preferably provides a single doseinjectable volume which is about 5-50 cc's and preferably about 15-30cc's.

Selection of the liquid introduced into the microbubble/hyperpolarized³He combination is important. As discussed above, the liquid 40 isselected such that it provides a relatively long T₁ for thehyperpolarized gas as the gas may exit the microbubble or contact thefluid as it attempts to diffuse through the walls of the microbubble.For in vivo applications, it is preferred that the injection liquid beselected so as to be non-toxic and non-depolarizing to thehyperpolarized gas. Preferably, the liquid will be selected such that ithas a low proton density along with the low solubility for ³He as notedabove. Preferably, the proton density is less than or equal to about 125mol/L, and more preferably less than about 120 mol/L, and still morepreferably less than about 115 mol/L. It is further preferred, forliquids which have a relatively high oxygen solubility value, that theliquid be processed to be more compatible with the hyperpolarized gas.For example, it is preferred that the liquid be at least partiallyde-oxygenated and/or partially de-ionized prior to introduction into thecontainer or transport vessel with the hyperpolarized gas. It is morepreferred that the liquid be sterilized and substantially de-oxygenatedand/or substantially de-ionized. Other modifications and treatmentprocesses can also be performed on the liquids to make them morepolarization-friendly. For example, certain elements of the liquids canbe substituted or deuterated and the like.

Of course, a plurality of liquids can also be used as the fluidcomponent, such as a liquid mixture or blend, whether miscible orimmiscible. Tests indicate that water is a suitable liquid (preferablydeoxygenated), as well as D₂O. Water is compatible and substantiallynon-depolarizing to ³He. Other liquid carriers are known such as thosedescribed in PCT/US97/05166 to Pines et al.

Previously, as noted in co-pending and co-assigned U.S. patentapplication Ser. No. 09/163,721, entitled Hyperpolarized Noble GasExtraction Methods, Masking Methods, And Associated TransportContainers, adding about 20 cubic centimeters of partially degassedwater into the chamber of a 250 ml container changed the associated T₁of the gas in the container from about 8 hours to about 5 hours. Thecontents of this application is hereby incorporated by reference as ifrecited in full herein.

For a microbubble mixture comprising deoxygenated water as the fillerwall 41, an estimation of the T₁ of the ³He in such a microbubblemixture can be described by equation 1.10. For the estimation, anestimate of the solubility of helium and the density of protons in thefluid is established. The solubility of helium in water as stated byWeathersby et al., in Solubility of inert gases in biological fluids andtissues, Undersea Biomedical Research 7(4), 277-296 (1980), is given as0.0098. The proton density of water is 111 mol/L (compared to 131.4 forLDPE). Thus, the ratio of water relaxivity to LDPE relaxivity is(0.0098/0.006) (111/131)^(1/2)=1.5. Knowing that the LDPE relaxivity isabout 0.0012 cm/min, the water relaxivity value is about 0.0018 cm/min.Thus, to obtain an estimate of T₁, the volume of the bubble is dividedby the surface area. For an 8 micron bubble, the V/A is about 2.7×10⁻⁴cmand T₁ is about 0.15 min (9 seconds). Doubling the diameter of thebubble to 16 micrometers can increase the time to 18 seconds. Using D₂Oas the fluid can provide a T₁ of about 36 seconds.

FIG. 6 is a block diagram of the preferred method of forming aninjectable ³He microbubble solution. A quantity of microbubbles isintroduced into a container (or gas holding chamber) (Block 100).Preferably the microbubbles are sized with a diameter which is about 10μm or less (Block 102). Next, a quantity of hyperpolarized ³He gas isintroduced into the container (Block 110). The pressure in the containeris increased to above atmospheric pressure (Block 120), preferably tobetween 2-10 atmospheres of pressure (Block 122). Of course, thepressure can be increased before the ³He is introduced or, morepreferably, subsequent to or concurrently with the introduction of the³He into the container.

The liquid (or liquid solution or mixture) is then introduced into thecontainer (Block 130), preferably after a predetermined lapsed “transit”time. Preferably, the liquid is pre-selected to have one or more of lowsolubility for ³He, a high diffusion coefficient for ³He, and, whereappropriate to be substantially de-oxygenated and/or de-ionized (Block132). The liquid can be limited in quantity (Block 134) and/or premixedwith another quantity of hyperpolarized gas (Block 136). The liquid, andthe microbubble/³He then combine or reside together to form theinjectable microbubble formulation (Block 140). Preferably, theinjectable formulation is sized in a deliverable bolus of less than orequal to about 50 cc's (Block 142).

Bubble Packing

It is preferred that the amount of liquid introduced into the chamberwith the microbubble/³He mixture be restricted to an amount about equalto or less than the volume of the combined volumes of ³He andmicrobubbles in order to pack the ³He within the “loaded” microbubble.As liquid volume decreases, signal strength based on same can increaseand less dilution of ambient ³He makes solubility appear smaller. Forexample, a 2 to 1 or 1 to 1 liquid to gas/microbubbles ratio or less,i.e., 20 cc's of microbubbles, 40 cc's ³He, and 60 cc's of liquid willprovide a 1—1 ratio.

Alternatively, or additionally, increased quantities of polarized ³Hecan be initially added to the liquid (premixed) to inhibit the tendencyof the ³He to migrate from the bubbles by providing at least residualamounts of ³He within the liquid itself. This can build up the quantityin the solution and reduce leaching from the microbubbles. Thisadditional or “surplus” ³He can be added to the liquid before orconcurrently with the liquid's introduction into the microbubble mixturein the container. For example, for a mixture comprising about 20 cc's ofmicrobubbles, 20 cc's of gas, and a liquid in an amount less than about40 cc's, a 20 cc amount of ³He can be introduced to the liquid (prior tointroduction into the container) to form the combined pre-mix liquidwhich is then directed into the chamber with the ³He and microbubbles.

Stated differently, the instant invention recognizes that the T₁ of thesolution is sensitive to bubble dilution in the liquid. Minimizing theliquid introduced into the mixture can minimize the equilibriumdifferential in the liquid mixture, which, in turn, should reduce theamount of depolarization occurring due to the leaching action. As such,a larger fraction of the ³He will remain within the bubble. Saturationcorresponds to solubility, which is a volume/volume measurement of about0.01 according to the present invention

Alternatively, or additionally, adding a liquid with previouslyintroduced quantities of helium gas (i.e., the premix solution) can alsoreduce the partial pressure difference in the combined mixture, whichcan also facilitate a larger fraction of the ³He to remain within thebubble.

These “bubble-packing” methods, particularly when used with alow-solubility liquid, can result in a higher T₁ formulation. Inaddition, using deuterated water for the solvent or liquid (or as acomponent thereof) can also help increase the T₁.

Due to the relatively short efficacy life of the injectable microbubble³He formulation, it is preferred that a rapid mixing and delivery systembe employed to administer the formulation to a subject temporallyrelated to initiation of the imaging sequence. That is, thepharmaceutical grade in vivo microbubble formulation is mixed onsite,temporally and physically proximate to or related to the point ofinjection, preferably mixed within about 30 seconds from the time ofinjection, and more preferably, rapidly and effectively mixed withinabout 10 seconds from the time of injection.

In any event, in operation, a measurement is preferably taken in advanceor concurrently with the injection via a NMR coil 31 on the injectioncontainer or delivery path (conduit, syringe body, etc.) toaffirm/determine the polarization level of the solution to allow thesignal intensity to be correlated with the polarized level of thehyperpolarized solution which is delivered.

As shown in FIG. 4, the injection mixture 45 is withdrawn from themixing chamber/transport container 30 into a syringe 70 (FIG. 5B) whichis positioned in a port or septum operably associated with the valve 50on the bottom of the container so that the liquid restricted mixture canbe easily removed (with the help of gravity). Valves 50, 51 are alsoemployed to control the pressure of the container. Typical valvesinclude Luer Lok™ valves, glass valves such as those available fromKonte Kimbles™, and polymer material valves can also be used as is knownby those of skill in the art. Of course, other extraction methods anddevices can also be used, such as those described in the co-pending andco-assigned patent application discussed above. Preferably, the syringe70 and any O-rings and valves positioned proximate thereto are formedfrom or coated with materials (at least the gas contacting surfaces)which are polarization-friendly as will be discussed further below.Further, the containers and syringes and other gas contacting devicesare preferably prepared to remove paramagnetic and magnetic impuritiesand oxygen and the like as will also be discussed further below. Inaddition, capillary stems and other separation or isolation means can beemployed to separate potentially depolarizing valve members from thepolarized gas as is discussed in co-pending and co-assigned U.S. patentapplication Ser. No. 09/334,400, the contents of which are herebyincorporated by reference as if recited in full herein.

Vacuum-Based Methods

In an alternative microbubble fabrication method, a vacuum typemicrobubble formulation method is employed. Referring to FIGS. 5A and5B, a quantity of microbubble shells 10 can be introduced under vacuumto an evacuated (cleaned/prepared) container 30. A quantity of gaseous³He 20 can be directed into the container (the vacuum pulls the gas intothe container). The evacuated state of the microbubbles induces the ³Hegas to rapidly enter and/or fill the microbubble shells. Next, asub-container 70 such as a syringe which is pre-filled with a liquidcarrier solution (such as deoxygenated fluid, liquid, or water) can beinjected into the container 30. The container 30 can be re-oriented toallow the subcontainer such as a syringe 70 to be backfilled with(preferably saturated) the bubble/polarized ³He/liquid solution. Asshown in FIG. 5B, the backfilled syringe can then be detached andinserted into a catheter positioned in the subject. Alternatively, aLUER LOK™ valve system can be operated to direct the solution downconduit into the catheter and thereby injected. In operation, the vacuumis preferably pulled to at least 50 microns (millitorr), and morepreferably to at least 10 microns. Thus, the evacuated method alsoallows for a relatively rapid or fast mix and delivery system.

Containers

Preferred materials for containers include aluminosilicates such asPyrex® or hyperpolarized gas contacting surfaces formed of materialsincluding non-magnetic high-purity metal films, high-purity metaloxides, high purity insulators or semi-conductors (such as high puritysilicon) and polymers. As used herein, “high purity” includes materialswhich have less than about 1 ppm ferrous or paramagnetic impurities andmore preferably less than about 1 ppb ferrous or paramagneticimpurities. Preferred polymers for use in the containers describedherein include materials which have a reduced solubility for thehyperpolarized gas. For the purposes of the inventions herein, the term“polymer” is to be broadly construed to include homopolymers,copolymers, terpolymers and the like and should also include blends andmixtures thereof. The terms “blends and mixtures thereof” include bothimmiscible and miscible blends and mixtures. Examples of suitablematerials include, but are not limited to, polyolefins (e.g.,polyethylenes, polypropylenes), polystyrenes, polymethacrylates,polyvinyls, polydienes, polyesters, polycarbonates, polyamides,polyimides, polynitriles, cellulose, cellulose derivatives and blendsand mixtures thereof. It is more preferred that the coating or surfaceof the container comprise a high-density polyethylene, polypropylene ofabout 50% crystallinity, polyvinylchloride, polyvinylflouride,polyamide, polyimide, or cellulose and blends and mixtures thereof. Seealso co-pending and co-assigned U.S. patent application Ser. No.09/334,400, the contents of which are hereby incorporated by referenceas if recited in full herein.

Of course, the polymers can be modified. For example, using halogen as asubstituent or putting the polymer in deuterated (or partiallydeuterated) form (replacement of hydrogen protons with deuterons) canreduce the relaxation rate. Methods of deuterating polymers are known inthe art. For example, the deuteration of hydrocarbon polymers isdescribed in U.S. Pat. Nos. 3,657,363, 3,966,781, and 4,914,160, thedisclosures of which are hereby incorporated by reference herein.Typically, these methods use catalytic substitution of deuterons forprotons. Preferred deuterated hydrocarbon polymers and copolymersinclude deuterated paraffins, polyolefins, and the like. Such polymersand copolymers and the like may also be cross-linked according to knownmethods.

It is further preferred that the polymer be substantially free ofparamagnetic contaminants or impurities such as color centers, freeelectrons, colorants, other degrading fillers and the like. Anyplasticizers or fillers used should be chosen to minimize magneticimpurities contacting or positioned proximate to the hyperpolarizednoble gas.

Alternately, in another embodiment, the contact surface can be formedfrom a high purity metal. The high purity metal can provideadvantageously low relaxivity/depolarization resistant surfaces relativeto hyperpolarized noble gases.

As noted above, any of these materials can be provided as a surfacecoating on an underlying substrate or formed as a material layer todefine a friendly contact surface. If used as a coating, the coating canbe applied by any number of techniques as will be appreciated by thoseof skill in the art (e.g., by solution coating, chemical vapordeposition, fusion bonding, powder sintering and the like). Hydrocarbongrease can also be used as a coating. The storage vessel or containercan be rigid or resilient. Rigid containers can be formed of Pyrex™glass, aluminum, plastic, PVC or the like. Resilient vessels arepreferably formed as collapsible bags, such as collapsible polymer ormetal film bags. Examples of materials which can provide oxygenresistance as well as low solubility include but are not limited to PET(polyethylene terphthalate), PVDC (polyvinylidene dichloride), Tedlar™(polyvinylfluoride), cellophane and polyacrylonitrile.

Preferably, care is taken to insure that all fittings, seals, and thelike which contact the hyperpolarized gas or which are locatedrelatively near thereto are manufactured from materials which arefriendly to polarization or which do not substantially degrade thepolarized state of the hyperpolarized gas. For example, as noted above,many commercially available seals include fluoropolymers or fillers andthe like which are not particularly good for the preservation of ³Hehyperpolarized gases because of the solubility of the material with thehyperpolarized gas.

Inasmuch as many common gasket materials are fluoropolymers or containundesirable fillers, they can potentially have a substantiallydepolarizing effect on the gas. This can be especially acute withrespect to ³He. This can be attributed to a relatively high solubilityof helium in most fluoropolymers due to the larger void space in thepolymer attributable to the large fluorine atoms. Indeed, preliminarytests indicate that materials of common O-rings (such as Viton™, Kel-F™,ethylene-propylene, Buna-N™, and silicone) exhibit far worse relaxationproperties than would be expected from the relaxation rate of purepolymers. Most conventional O-rings are so depolarizing that they candominate the relaxation of an entire hyperpolarized gas chamber. Indeed,commercial ethylene propylene O-rings exhibit ⅓-½ the relaxation timecompared to pure LDPE with ¹²⁹Xe. The faster relaxation rate can beexplained because magnetic impurities in the O-rings can be introducedby such things as colorants and fillers and the like. Therefore, it ispreferred that the containers of the present invention employ seals,O-rings, gaskets and the like with substantially pure (substantiallywithout magnetic impurities) hydrocarbon materials such as thosecontaining polyolefins. Examples of suitable polyolefins includepolyethylene, polypropylene, copolymers and blends thereof which havebeen modified to minimize the amount of magnetically impure fillers usedtherein. Additional suitable seals include hydrocarbon grease andhydrocarbon gaskets and O-rings made from polyethylene and the like.Thus, if a valve is used to contain the gas in the chamber 30, it ispreferably configured with reduced magnetic impurities (at least thesurface) O-ring and/or with hydrocarbon grease. Of course, becausefillers and plasticizers are employed, then it is preferred that they beselected to minimize the magnetic impurities, one preferred materialbeing substantially pure carbon black.

In an alternative embodiment, the O-ring seal can be configured with theexposed surface coated with a high purity metal as discussed for thecontainer surface. Similarly, the O-ring or seal can be coated or formedwith an outer exposed layer of a polymer at least “L_(p)” thick. Forexample, a layer of pure polyethylene can be positioned over acommercially available O-ring. One preferred commercially availableO-ring material for ¹²⁹Xe is a Teflon™ coated rubber O-ring or alow-relaxivity polymer as discussed above. The void spaces in Teflon™(although it is a fluoropolymer) do not affect ¹²⁹Xe as much as they do³He because ¹²⁹Xe is much larger than fluorine, which is much largerthan ³He. As discussed previously, fluoropolymers can be used as sealswith ¹²⁹Xe but are not preferable for use with arrangements where theseal may contact the hyperpolarized ³He.

In order to determine the “L_(p)” thickness, wherein the layer thickness(“L_(th)”) is at least as thick as the polarization decay length scale(“L_(p)”), one can calculate or determine the thickness for a particularmaterial type according to the equation:

L _(p) ={square root over (T_(p)D_(p))}  (6)

where T_(p) is the noble gas nuclear spin relaxation time in the polymerand D_(p) is the noble gas diffusion coefficient in the polymer. Forexample, a layer of substantially pure polyethylene can be positionedover a commercially available O-ring. Alternatively, the O-ring or sealcan be coated with a surface material such as LDPE or deuterated HDPE orother low-relaxivity property material. It is also preferred that therelaxivity value “Υ” is less than about 0.0012 cm/min for ³He.

When bags with long surface relaxation times are used as containers,other relaxation mechanisms can become important. One of the mostimportant additional relaxation mechanisms is due to collisions of thenoble gas with paramagnetic oxygen. Because O₂ has a magnetic moment, itcan relax hyperpolarized gases in the same manner as protons. Given thisproblem, care should be taken to reduce the oxygen content in thestorage container through careful preconditioning of the container, suchas by repeated evacuation and pure gas purging procedures. Preferably,the container is processed such that the O₂ concentration yields a T₁ ofabout 1000 hours or more. More preferably, the container is processed toobtain an O₂ concentration on the order of about 6.3×10⁻⁶ atm or less orabout 10⁻⁷ atm or less, and even more preferably less than about 1×10⁻¹⁰atm. Additionally, the evacuation/purge procedures can include heatingthe container or other evacuating or pumping methods to additionallyfacilitate the removal of any remaining (monolayer) residual amounts ofmoisture or water.

Preferably, the mixing container/interfaces, syringes, and tubing areprepared in advance of use to minimize any preparation required at thetime of use at the gas injection site. Therefore, preferredpre-conditioning or equipment preparation methods such as cleaning,evacuating, and purging the components to remove oxygen and paramagneticcontaminants are preferably done off-site. Afterpreparation/conditioning, the pre-conditioned syringes can be stored atthe hospital for use under pressure with a noble gas or benign liquidtherein. This pre-filled gas or fluid storage can minimize the potentialfor the containers, syringes or components to degas (gas from the matrixof a material such as oxygen can migrate into the chamber onto thecontact surfaces), and can also minimize air leaking into the container.Alternatively, or in addition to the pre-conditioning, the pressurizedtubing and delivery vessels (and/or syringes) can be sealed with checkvalves or other valved ports. In another alternative, vacuum tightvalves can allow the tubes and containers to be stored for use undervacuum rather than under positive pressure.

The hyperpolarized gas is collected (as well as stored, transported, andpreferably delivered) in the presence of a magnetic field. For ³He, themagnetic field is preferably on the order of at least 5-30 gaussalthough, again, higher (homogeneous) fields can be used. The magneticfield can be provided by electrical or permanent magnets. In oneembodiment, the magnetic field is provided by a plurality of permanentmagnets positioned about a magnetic yoke which is positioned adjacentthe collected hyperpolarized gas. Preferably, the magnetic field ishomogeneously maintained around the hyperpolarized gas to minimize fieldinduced degradation.

In operation, the injected hyperpolarized ³He of the present inventioncan provide signal strengths even in relatively small quantities whichcan be detected by known NMR spectroscopy and imaging methods. In apreferred embodiment, a second quantity of ³He is delivered viainhalation to allow both a perfusion (injection based) and ventilation(inhalation based) MR image or “VQ scan”. Because ³He is used for bothexcitations/data acquisition, for vasculature images, a single NMRexcitation coil (chest coil) can be conveniently used to obtain bothsignals.

Dissolving Xenon

In addition, microbubbles can be used as a dissolving mechanism toassist in dissolving xenon into a liquid, which has traditionally beenextremely reluctant to dissolve into a carrier liquid. As an example, avial with a plurality of microbubbles is provided. Next, polarized ¹²⁹Xeis added to the vial. A solvent or liquid mixture (preferablyphysiologically compatible and non-toxic and sterile) is added toprovide an optimal bubble packing fraction (i.e., a limited amount ofliquid as discussed above). Alternatively, the amount of liquid can beincreased to provide a diluted liquid mixture, thus inducing the xenonto exit the bubble to achieve equilibrium. In any event, the xenonrapidly leaches into the solution out of the microbubble. Preferably,prior to injection, the bubbles are strained or filtered out leaving aliquid with dissolved xenon. Advantageously, the microbubbles can thenact as an accelerant to assist in the xenon dissolving in a liquid,which, in the past has been time consuming and problematic.

Of course, because the microbubbles will preferably be filtered from thedissolved xenon, the microbubble size is not limited by the injectionthereof into in vivo systems. Exemplary compatible fluids are describedin PCT/US97/05166 to Pines et al.

Drug Evaluations

Although it is preferred that the microbubble injectable formulation bea pharmaceutical grade in vivo formulation (such as a non-toxic andsterilized solution, with the alkali metal separated from thehyperpolarized gas according to FDA standards(for alkali spin exchangehyperpolarized gases), the present invention is not limited thereto.Indeed, rapid advances are being made with the ability to treat andtarget many diseases with innovative drug and drug therapies. NMRspectroscopy based on hyperpolarized gases can be used to observe theeffects of administered drugs on the biochemistry of the organism or thechanges in the drug which occur following its administration. Theinstant invention can allow for improved sensitivity and potentiallyhigher resolution information for evaluations of the treatments or eventhe chemical processes underlying a disease state with respect to thedesired target tissues or organs within the body. For example, deliveryof the injectable microbubble solution to an animal or in vitro targetcan evaluate the efficacy of treatment on function or the progression orregression/improvement of a condition in the pulmonary vasculature,cardiac, brain, or other tissue, organ, or system.

The foregoing is illustrative of the present invention and is not to beconstrued as limiting thereof. Although a few exemplary embodiments ofthis invention have been described, those skilled in the art willreadily appreciate that many modifications are possible in the exemplaryembodiments without materially departing from the novel teachings andadvantages of this invention. Accordingly, all such modifications areintended to be included within the scope of this invention as defined inthe claims. In the claims, means-plus-function clauses are intended tocover the structures described herein as performing the recited functionand not only structural equivalents but also equivalent structures.Therefore, it is to be understood that the foregoing is illustrative ofthe present invention and is not to be construed as limited to thespecific embodiments disclosed, and that modifications to the disclosedembodiments, as well as other embodiments, are intended to be included.

That which is claimed is:
 1. A method of preparing and subsequentlyinjecting polarized gaseous ³He in vivo, comprising the steps of: (a)introducing a quantity of microbubbles into a container; (b) applying avacuum to the container; (c) directing a first quantity ofhyperpolarized ³He gas into the evacuated container with themicrobubbles; (d) directing a second quantity of a fluid into thecontainer to form a bubble solution after the first directing step; and(e) injecting the bubble solution into a subject.
 2. A method accordingto claim 1, wherein the second quantity of fluid comprises asubstantially deoxygenated fluid.
 3. A method according to claim 1,wherein said injecting step includes delivering the bubble solution to acatheter inserted into the vein of a subject.
 4. A method according toclaim 1, wherein steps (b) through (d) are carried out temporallyproximate to step (e).
 5. A method according to claim 1, wherein steps(b) through (e) are carried out in less than about 30 seconds.
 6. Amethod according to claim 1, further comprising the step of acquiringNMR signal data based on the detection of the hyperpolarized gasdelivered to a target via the bubble solution.
 7. A method according toclaim 1, wherein the second quantity of fluid is a liquid.
 8. A methodaccording to claim 7, wherein the bubble solution is an injectableformulation which is physiologically compatible.
 9. A method accordingto claim 7, wherein the liquid is nontoxic and sterile.
 10. A methodaccording to claim 7, wherein the liquid is added in an amount which isgreater than the combined volumes of microbubbles and gaseous ³He.
 11. Amethod according to claim 1, wherein the chamber is held in an evacuatedstate while the microbubbles are directed therein.
 12. A methodaccording to claim 11, the chamber remains in an evacuated state whilethe polarized ³He is directed therein.
 13. A method according to claim1, wherein said applying step decreases the pressure in the chamber toless than about 50 microns (millitorr).
 14. A method according to claim13, wherein the pressure is decreased in the chamber to at least about10 microns.
 15. A method of preparing polarized gaseous ³He, comprisingthe steps of: (a) evacuating a container; (b) introducing a quantity ofmicrobubbles into the evacuated container; (c) directing a firstquantity of hyperpolarized ³He gas into the evacuated container with themicrobubbles after the microbubbles are introduced in the evacuatedcontainer; and (d) subsequently directing a second quantity of a fluidinto the container to form a bubble solution.
 16. A method according toclaim 15, wherein the second quantity of fluid is a substantiallydeoxygenated liquid.
 17. A method according to claim 15, wherein saidbubble solution is a biocompatible injectable formulation for in vivoadministration.
 18. A method according to claim 15, wherein the bubblesolution is formulated for delivery to an in vitro target.
 19. A methodaccording to claim 13, further comprising the steps of delivering thebubble solution to a target and acquiring NMR signal data based on thedetection of the hyperpolarized gas delivered to a target via the bubblesolution.
 20. A method according to claim 16, wherein the liquid issubstantially deoxygenated water.
 21. A method according to claim 15,wherein the second quantity of fluid is a liquid which is substantiallyde-ionized.
 22. A method according to claim 15, wherein the secondquantity of fluid is a liquid which comprises an increased concentrationof D₂O relative to that naturally found in water.
 23. A method accordingto claim 15, wherein the fluid is a liquid that has a low protondensity.
 24. A method according to claim 15, wherein the microbubblematerial has a high diffusion coefficient for ³He.
 25. A methodaccording to claim 15, further comprising the steps of injecting thebubble solution into a biological subject and measuring the polarizationlevel of the ³He proximate in time to said injecting step, said step ofdirecting the hyperpolarized ³He into the evacuated container is carriedout temporally proximate to said injecting step.
 26. A method accordingto claim 15, wherein the bubble solution is an injectable formulationwhich is sized in the range of over 7 cc's and at or below about 50cc's.
 27. A method according to claim 15, further comprising the step ofmeasuring the polarization of the bubble solution post-formulation andproximate to delivery to a target to determine level of polarizationthereat.
 28. A method according to claim 16, wherein the second quantityof fluid is a liquid in an amount which is greater than the combinedvolumes of microbubbles and gaseous ³He.
 29. A method according to claim7, wherein the liquid has a low solubility for ³He.
 30. A methodaccording to claim 15, wherein the fluid is a liquid that has a lowsolubility for ³He.
 31. A method according to claim 7, wherein themicrobubble material has a high diffusion coefficient for ³He.
 32. Amethod according to claim 7, wherein the injectable formulation is sizedin the range of above 7 cc's and at or below about 50 cc's.
 33. A methodaccording to claim 32, wherein the injectable formulation is sized inthe range of between above 7 cc's to about 15 cc's.
 34. A methodaccording to claim 7, wherein the quantity of hyperpolarized ³He in thechamber is larger than the quantity of liquid.
 35. A method according toclaim 7, wherein the quantity of hyperpolarized ³He in the chamber is atleast about two times greater than the quantity of liquid.
 36. A methodaccording to claim 15, wherein the fluid is a liquid and the bubblesolution is an injectable formulation sized in the range of above 7 cc'sand at or below about 50 cc's.
 37. A method according to claim 36,wherein the injectable formulation is sized in the range of betweenabove 7 cc's to about 15 cc's.
 38. A method according to claim 15,wherein the fluid is a liquid and wherein the quantity of hyperpolarized³He in the chamber is larger than the quantity of liquid.
 39. A methodaccording to claim 15, wherein the fluid is a liquid and wherein thequantity of hyperpolarized ³He in the chamber is at least about twotimes greater than the quantity of liquid.
 40. A method according toclaim 1, wherein the second quantity of fluid is a liquid that is heldin a syringe prior to introducing the second quantity into thecontainer.
 41. A method according to claim 40, wherein the syringe ispre-filled with the liquid.